Team:UCLA/Notebook/Honeybee Silk/5 May 2015

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Colony PCR of 5/4 Transformation

5/4 Transformation Results

• Individual colonies were present on 1:1 plates, none/indiscernible present on 1:10 plates.
• We picked 3 colonies from each the chloramphenicol plates (silk and silk+promoter) and suspended each in an epi tube with 99 uL ddH20.

Colony PCR Reaction

• using Q5, transformed E. coli, and the VF2 and VR primers in preparation for insertion into psb1c3
• Three 25 uL reactions each for:
  • Silk with VF2 and VR
  • SIlk+promoter with VF2 and VR

Component	Volume (out of 25uL)
5X Q5 Reaction Buffer*	5uL
10mM dNTPS*	0.5uL
10mM VF2 primer*	1.25uL
10mM VR primer*	1.25uL
Transformed cells in ddH2O	1uL
Q5 High Fidelity DNA Polymerase	0.25uL
Nuclease Free Water*	15.75uL

• I made a Mastermix of the starred components, then added 23.75uL of the mix to each PCR strip, followed by the corresponding transformed cells and Q5 DNA Polymerase.

Step	Temperature	Time
Initial Denaturation	98C	3 min
Cycles (x25)	98C	10s
Annealing	66C	15s
Extension	72C	15s
Final Extension	72C	2min
Hold	12C	Hold

• TM calculated using NEB TM Calculator
• Total Run Time 35 minutes (including ramp times)

Gel Visualization of PCR Products

• 5uL of 6X loading dye was added to each 25uL PCR product
• 10uL of 1kb ladder was prepared with 2uL of 6X loading dye

I ran two 5uL samples of each PCR product against the ladder, one well was left empty between the different samples.

From left to right: Ladder, S1, S2, S3, SP1, SP2, SP3 Expected Band sizes: ~1200bp, ~1500bp File:Gel 5/5.tif

Inoculation

99uL of each sample was inoculated in 6 difference culture tubes containing 10mL LM and 1uL of 1000x chloramphenicol and incubated overnight at 37C.